Disulfide bond-targeted radiolabeling: tumor specificity of a streptavidin-biotinylated monoclonal antibody complex.
A site-specific labeling method was developed in which sulfhydryl groups of a murine IgG2a anti-ovarian monoclonal antibody, 5G6.4, were biotinylated with N-iodoacetyl-N'-biotinylhexylenediamine (Compound 1) following partial reduction of disulfide bonds with dithiothreitol. Reaction of 1-alkylated 5G6.4 with 125I-streptavidin gave immunoreactive streptavidin-1-biotinylated complexes. Radio-fast protein liquid chromatography data were consistent with the formation of a stable monovalent streptavidin-half-antibody complex as the major species. In vivo specific localization of these radioantibody conjugates to human tumor xenografts of ovarian carcinoma was confirmed by a comparative biodistribution study in nude mice using as a control the nonspecific 125I-streptavidin-1-alkylated UPC-10 (an irrelevant IgG2a monoclonal antibody) complex prepared analogously as described above. Tumor uptake for radiolabeled 5G6.4 [0.279 +/- 0.041% (SE) kg injection dose/g) was significantly greater [P less than 0.025] than for UPC-10 [0.165 +/- 0.027% kg injection dose/g]. The tumor:blood ratio (7.38 +/- 1.285) for 5G6.4 was approximately 3 times that for UPC-10 (2.48 +/- 0.708, P less than 0.01). This sulfhydryl site-directed approach demonstrated that reduced disulfides of monoclonal antibodies are viable sites for attaching labels without significant loss of in vitro and in vivo immunoreactivity.
del Rosario
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RB
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor 48109-0028.
Wahl
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RL
D000818:Animals; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D001710:Biotin; D004220:Disulfides; D006801:Humans; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D051379:Mice; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000175:diagnosis*; D019809:Streptavidin
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D004220:Disulfides; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D001710:Biotin; D019809:Streptavidin